Taqman荧光定量RT—PCR在呼吸道感染患者临床标本非SARS冠状病毒检测中的应用

目的建立检测四种常见人非SARS冠状病毒核酸特异的快速、敏感的TaqManqRT—PCR检测方法，应用于急性呼吸道感染患儿的感染分析。方法分别应用TaqManqRT—PCR与普通RT—PCR平行检测248份呼吸道标本，对方法的灵敏性、特异性和稳定性以及临床标本的适用性进行比较评价，阳性标本以体外转录RNA为标准品进行病毒载量定量。结果本方法可对HKU1、NL63、229E、OC43四种冠状病毒进行特异性诊断，与其他病毒无交叉反应，检测灵敏度可达10拷贝／μl，检测线性范围可达10^1～10^8拷贝／μl，248份标本中HKU1、NL63、229E、OC43阳性率依次为1．2％，0．8％，1．2％，1．6％，其中OC43荧光RT—PCR法检出率高于普通RT—PCR，其余三种病毒两种方法检测结果一致。非SARS冠状病毒阳性标本均检出于12月至次年5月。结论建立的TaqManRealtimeRT-PCR法具有特异性强、灵敏性高的特点，是开展非SARS冠状病毒的临床检测与疾病监测的有效技术手段。

Detection of human coronaviruses non SARS in clinical specimens from patients with respiratory tract infection by use of real-time reverse-transcriptase polymerase chain reaction with taqman probes

Objective To develop a sensitive specific and rapid real-time quantitative polymerase chain reaction （PCR） assay using Taqman probe for detecting four common human coronavirus （HCoV） excluding SARS. To detect four eoronaviruses in children with acute respiratory infection use the assay. Methods We designed and obtained specific pairs of primers and Taqman probes sequences to identify type-specific conserved regions of the four viruses. Simultaneous Detection of the four HCoV （ non SARS） by using fluorescent real time PCR and common real time PCR respectively for 248 samples. The specificity, sensitivity and stability of the assay were evaluated. Results The detection ability for NL63, HKU1, 229E by using the designed assays were the same as common RT-PCR, but for OC43 the assays demonstrates higher sensitivity compared with common RT-PCR. No cross-reaction with the other examined RNA viruses was observed. The detection limits were up to 10 eopies/μl and this assay allowed quantitation of four viruses over a range of 101 to 10s RNA copies/μl. These results and the ability to detect virus in sample by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay, viruses positive samples were all examed in December till May of next year. Conclusions The results showed that the assay had high sensitivity and specificity. TaqMan RT-PCR have proven useful in assisting scientists in respiratory tract and it is an effective way for clinical examination and surveillance of coronavirus.